COMT is an enzyme which is present in many tissues in the human body. Its primary function is to inactivate and thus facilitate the removal from the body of adrenaline, noradrenaline and dopamine (catecholamines), which act as hormones and neurotransmitters. The present inventors have shown previously that the cellular levels of the enzyme are regulated by the female hormone, estradiol. Many chemicals, both man-made and naturally occurring, can mimic or inhibit the actions of estradiol. If introduced in humans, such chemicals may cause disruption of the normal actions of estradiol and cause health problems at all stages of human life. These chemicals, together with others that affect the actions and metabolism of other hormones, are called collectively endocrine disruptors. Chemicals which affect estradiol actions are probably the most important class of endocrine disruptors. They have been associated with falling sperm counts in Western males, and in the causation of common cancers such as breast cancer and ovarian cancer.
The present inventors have shown that polychlorinated biphenyls (PCBs; universally recognized as posing very serious threats to human health and the wider environment) and a variety of plasticisers and related chemicals are able to modulate levels of COMT protein expression. None of these chemicals is closely related to estradiol, but nevertheless they modulate levels of COMT via the same mechanism as estradiol (Ho et al., 2008a,b). Thus, an assay of COMT protein in cells exposed to such a chemical is a measure of the estrogenic activity of the chemical. Large numbers of chemicals present in, or potentially to be introduced into, the environment have never been assessed for their endocrine disrupting estrogenic potential. This is because the two-generation rat test, which is the recognized test for endocrine disruption, is very time-consuming and extremely expensive. Legislation enacted by the European Union indicates that there are approximately 30,000 chemicals which must be tested. As stated above, assay of COMT protein potentially provides a measure of estrogenic endocrine disrupting potential. Current methods of assaying COMT protein are slow, labor-intensive and limited in sample handling capacity. For example, current assay of human COMT protein concentration is accomplished by use of polyacrylamide electrophoretic (SDS-PAGE)/Western blotting assays. SDS-PAGE involves the use of buffers and gels containing acrylamide and bisacrylamide, which are neurotoxic, and sodium dodecyl sulphate which is a lung irritant and sensitizer. Although many laboratories use commercially available pre-cast gels and prepared buffers to minimize staff exposure to these chemicals, they are, nevertheless, within the laboratory environment and require appropriate safety procedures to be carried out to ensure safe handling and disposal. SDS-PAGE/Western blotting is time-consuming, laborious, difficult to quantify accurately, relatively limited in its capacity for processing samples and consequently is expensive.
An alternative method of estimation COMT concentration is to assay COMT activity and extrapolate the results. This involves use of radioactive substrates for the enzyme, with their attendant hazards and problems of disposal of contaminated materials. Accordingly, there is a need for a COMT assay that is simple, inexpensive, and can handle many samples. The assay described below addresses these problems, because it is simple to use, inexpensive, and has a high sample handling capacity. Thus, it is particularly well-suited as an initial screening system for potential estrogenic endocrine disrupters.